Activity of the endogenous RNA polymerase on fixed polytene chromosomes.

Fixed polytene chromosomes can serve as templates for RNA synthesis in situ, using the endogenous chromosomal DNA-dependent RNA polymerase. Labelling is mainly localized in band regions. However, radioactivity can also be found in interbands and puffs similar to that which occurs in vivo. It is also found by this technique that the nucleolar RNA polymerase appears to be active in these preparations and requires Mg²⁺ for activity. Since the pattern of the RNA transcribed in situ with the DNA-dependent RNA polymerase from E. coli of native chromosomes differs from that with endogenous RNA polymerase and resembles the one obtained with heat-treated chromosomes, it is suggested that the polymerase from E. coli does not act specifically on eukaryotic chromosomes.     

Quantitative conservation of chromatin-bound RNA polymerases I and II in mitosis. Implications for chromosome structure

RNA synthesis almost ceases in mitosis. It is ambiguous whether this temporal, negative control of RNA synthesis is solely because of the nature of chromosomes per se, (i.e., their condensed state), or to a physical loss of RNA polymerases along with other nuclear proteins which have been shown to pass into the cytoplasm in mitosis, or to their combined feature. Aside from such regulatory considerations, a question has also been raised as to whether RNA polymerases are constituents of metaphase chromosomes. To clarify these aspects of RNA polymerase-chromatin interaction in mitosis, the enzymes in chromosomes were quantitated and their levels compared to those in interphase nuclei and cells at various phases of the cell cycle. The results show that the amounts of form I, form II, and probably form III enzymes bound to a genome-equivalent of chromatin stay constant during the cell cycle. Thus, the mechanism for the negative control of RNA synthesis in mitosis appears to exist in the chromosomes per se, but not to be directly related to the RNA polymerase levels. This quantitative conservation of chromatin-bound RNA polymerases implies that they may persist as structural components of the chromosomes in mitosis.     

RNA SYNTHESIS BY EXOGENOUS RNA POLYMERASE ON CYTOLOGICAL PREPARATIONS OF CHROMOSOMES

Cytological preparations of Drosophila polytene chromosomes serve as templates for RNA synthesis carried out by exogenous RNA polymerase (Escherichia coli). Incorporation of labeled ribonucleoside triphosphates into RNA may be observed directly by autoradiography. Because of the effects of rifampicin, actinomycin D, ribonuclease, high salt, and the requirement for all four nucleoside triphosphates, we conclude that the labeling observed over chromosomes is due to DNA-dependent RNA polymerase activity. Using this method, one can observe RNA synthesis in vitro on specific chromosome regions due to the activity of exogenous RNA polymerase. We find that much of the RNA synthesis in this system occurs on DNA sequences which appear to be in a nondenatured state.     

Chromosomal RNA synthesis in polytene chromosomes of Chironomus tentans

The presence of heterogeneous RNA of high molecular weight has been demonstrated on the giant chromosomes, in the nuclear sap and in the cytoplasm of the salivary glands in Chironomus tentans. The kinetic properties of this heterogeneous RNA have also been outlined in some detail. — Salivary glands were incubated for different time intervals (20, 45 and 180 min) in haemolymph, supplemented with tritiated cytidine and uridine. The different cellular components were isolated by micromanipulation and RNA extracted with an SDS-pronase solution and analysed with electrophoresis in agarose. — Heterogeneous, high molecular weight RNA with a peak around 35 S was saturated with label on chromosome I, II and III in 45 min, although the synthetic capacity was unchanged during at least 180 min incubation. This indicated a complete turnover of heterogeneous RNA on the chromosomes in less than 45 min. The turnover time in the giant puffs (the so called Balbiani rings) on the fourth chromosome, was even shorter and estimated to less than 30 min. No shift in the electrophoretic pattern of this heterogeneous RNA was found to occur on the chromosomes during long incubation times or during actinomycin D experiments. These labelling characteristics of heterogeneous RNA on the chromosomes indicate that all the different molecules in the heterogeneous RNA have a similar and rapid turnover. A conversion to smaller, stable molecules was excluded. — Heterogeneous RNA of a distribution corresponding to that on the chromosomes was found in the nuclear sap and also in the cytoplasm. The activity in both these cellular compartments increased between 45 and 180 min incubation. The distribution pattern for high molecular weight RNA was in all experiments similar on the chromosomes, in the nuclear sap and in the cytoplasm. It appears that at least a considerable part of the high molecular weight RNA leaves the chromosomes to enter the nuclear sap and lateron to some extent the cytoplasm in this high molecular form. Stable molecules of smaller size (6–15 S) did not appear during 180 min incubation. The data indicate, however, also a substantial breakdown of heterogeneous RNA to acid soluble products during this time.     

Tetrahymena telomerase RNA levels increase during macronuclear development.

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000-40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2- to 5-fold at 9-15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells.     

Migration of newly synthesized RNA during mitosis : III. Nuclear RNA in the cytoplasm of metaphase cells

It was shown by autoradiography in previous papers that RNA which is synthesized before mitosis and located in the nuclei, enters the cytoplasm at the onset of mitosis and returns to the nuclei of the daughter cells after mitosis. In order to study thenature of this migrating RNA we performed a sedimentation analysis of RNA isolated from the cytoplasm and chromosomes (nuclei) of metaphase and interphase cells in the synchronized culture of the Chinese hamster. Whereas the cytoplasm of interphase cells is found to contain RNA with sedimentation constants not higher than 28S, the cytoplasm of metaphase cells includes precursors of ribosomal and messenger RNA with sedimentation constants 32S, 45S and even higher. This means that RNA migrating from nuclei to cytoplasm during cell division retains its nuclear character. It is suggested that this property provides for the return of RNA synthesized before mitosis to the nuclei of the daughter cells.     

Intracellular distribution of low molecular weight RNA in Chironomus tentans

Low molecular weight RNA species are described in isolated nuclear components and cytoplasm of salivary gland cells of Chironomus tentans. In addition to 4S and 5S RNA and RNA in the 4–5S range previously described, at least three other components in the range below 16S are present. RNA, the molecular weight of which was estimated to 2.3 x 10⁵ and designed 10S RNA, can be observed only in nucleoli; other RNA, the molecular weight of which was estimated to 1.3 x 10⁵ and designed 8S RNA, was detected in the chromosomes, the nuclear sap, and the cytoplasm but not in the nucleoli; and a third type of RNA, the molecular weight of which was estimated to 8.5 x 10⁴ and designed 7S RNA, was present in nucleoli, chromosomes, nuclear sap, and cytoplasm. The substituted benzimidazole, 5,6-dichloro-1 (β-D-ribofuranosyl)benzimidazole (DRB), which gives a differential inhibition of the labeling of heterodisperse, mainly high molecular weight RNA in the chromosomes, does not inhibit the labeling of 8S RNA. The relative amounts of label in 8S RNA and 4–5S RNA (including 4S RNA and 5S RNA) in different isolated chromosomes, are distributed in proportion to the chromosomal DNA contents. The 8S RNA as well as the 7S RNA show a relative accumulation in chromosomes and nuclear sap with prolonged incubation time and are in this respect similar to intranuclear low molecular weight RNA species described by previous workers. Our data suggest, however, that these two types of RNA may differ in an important aspect from the previously described types since they are also present in the cytoplasm.     

Conjugation of polytene chromosomes in intraspecies hybrids of the virilis group of Drosophila. II. Hybridization of complementary RNA with polytene chromosomes in specimens]

A cytological hybridization of H-3-complementary RNA synthetized from DNA a template of D. virilis with the polytene chromosomes of D. virilis and the hybrids between D. virilis and D. texana, was carried out in situ. The uridine label of RNA was shown to be located mainly over the disc of the polytene chromosomes, the silver grains in interspecies hybrids being located over both homogous chromosomes including the unpaired gions.     

Localization of chromosomal RNA in human G-banded metaphase chromosomes

Chromosomal RNA with an 11% dihydropyrimidine content was extracted from human placental chromatin. Under appropriate conditions, this RNA showed wide-spread in situ hybridization to metaphase chromosomes. This included preferential hybridization to the telomeric regions and heterochromatic short arms of acrocentric chromosomes as well as significant hybridization to Q- and G-positive bands.     

Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster.

In situ hybridization of ¹²⁵I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ. This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead.     

The site of 5S RNA genes in human chromosome 1.

The major site of genes for human 5S RNA is in the long arm of chromosome 1. We find no evidence of sites in other chromosomes; if such exist, they are much smaller than the site in 1q.     

The location of 5S RNA genes in lampbrush polytene chromosomes from Drosophila

Drosophila polytene chromosomes were transformed into lampbrush-like structures by exposure to solutions of alkali-urea. In this process, the chromosomes shorten and widen, and the bands (chromomeres) extend laterally into loops leaving a central core between the paired homologues. The expanded polytene chromosomes are very similar in appearance to the true lampbrush chromosomes of amphibian oocytes and to ordinary chromosomes in pachytene. The denaturing effects of alkali-urea were partially counteracted by return of the treated chromosomes to Ringer solution. These observations are interpreted in terms of recent findings on protein backbones in chromosomes, and indicate that chromosomes generally may have very similar basic organization, despite differences due to species, polyteny and degree of condensation. To gain more information on the specific location of a structural gene, ¹²⁵I-labelled low molecular weight (containing 5S RNA) was hybridized in situ to normal and lampbrush-like polytene chromosomes. Autoradiography showed silver grain distribution for 5S RNA consistent with hybridization primarily to the loop regions of the lampbrush chromosomes rather than the core. This provides further indirect evidence that structural genes like 5S RNA may be located on the bands (chromomeres) and not the interbands of normal polytene chromosomes.     

Localization of 5S RNA and rRNA genes in the Norway rat

The genes coding for the two classes of ribosomal RNA molecules, 5S RNA and 18+28S RNA, have been localized in the Norway rat (Rattus norvegicus). The 18+28S RNA cistrons are found on three chromosomes, at secondary constrictions on the short arms of chromosomes 3 and 12 and at the telomere of the short arm of chromosome 11. These sites were confirmed using the silver staining technique for nucleolar organizer regions. Two sites were found for the 5S RNA genes; one is closely linked to the 18+28S gene site on chromosome 12. The second site is at or near the telomere of the long arm of chromosome 19.     

In vitro RNA synthesis in oocyte nuclei of the newt Notophthalmus

An incubation medium is described which supports RNA synthesis in isolated oocyte nuclei of the newt Notophthalmus, and which permits subsequent autoradiographic examination of the lampbrush chromosomes and nucleoli. By using different concentrations of α-amanitin we distinguish RNA synthesis due to RNA polymerases I, II and III. All RNA synthesis on loops is inhibited by 0.5 μ/ml of α-amanitin and is therefore due to polymerase II. Polymerase III is responsible for RNA synthesis at a small number of discrete sites in condensed chromatm. These include the centromere bars of three of four chromosomes, which probably represent 5S RNA synthesis, as well as 15–20 lesser sites scattered elsewhere. Polymerase I activity is confined to the nucleoli. Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Localization of 5 S RNA genes in Chironomus tentans

The genes for 5 S RNA in Chironomus tentans have been located to region 2A of chromosome II by cytological hybridization. RNA from individual chromosomes, nuclear sap and nucleoli of salivary gland cells hybridized with the identified 5 S RNA genes in region 2A of chromosome II. The results suggest a common origin of 5 S RNA in these different nuclear compartments.